Reversibly protected triazabutadienes

ABSTRACT

Triazabutadiene molecules and methods of use of triazabutadiene molecules, for example methods and compositions for yielding an aryl diazonium species from a triazabutadiene molecule, e.g., a protected aryl diazonium species in the form of a triazabutadiene. In some embodiments, an enzyme catalyzes the reaction yielding the aryl diazonium species from the triazabutadiene molecule. As an example, the methods and compositions herein may be used for delivery of drugs.

CROSS REFERENCE

This application is continuation-in-part of PCT Patent Application No.PCT/US18/22046 filed Mar. 12, 2018, which claims benefit of U.S.Provisional Patent Application No. 62/469,677 filed Mar. 10, 2017, thespecifications of which are incorporated herein in their entirety byreference.

This application is a continuation-in-part and claims benefit of U.S.patent application Ser. No. 15/751,555 filed Feb. 9, 2018, which is a371 application of PCT Patent Application No. PCT/US16/46624 filed Aug.11, 2016, which claims benefit of U.S. Provisional Patent ApplicationNo. 62/203,667 filed Aug. 11, 2015 and U.S. Provisional PatentApplication No. 62/203,725 filed Aug. 11, 2015, the specifications ofwhich are incorporated herein in their entirety by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 1552568awarded by NSF. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to protected aryl diazonium species thatcan be selectively released using enzymes.

BACKGROUND OF THE INVENTION

Degradation of triazabutadiene molecules to their respective diazoniumspecies is triggered by a protonation event on the N3 nitrogen atom(farthest from the aryl ring). In an effort to understand the reactivityof triazabutadienes, Fanghanel et al. (J. Prakt. Chem. 1977,319:813-826) showed that an N1 alkylated triazabutadiene molecule wasstable in acidic conditions. However, alkylation of these compounds iseffectively irreversible.

Inventors have surprisingly discovered that carboxylation on N1 oftriazabutadienes reversibly yields stabilized or protected versions oftriazabutadienes, e.g., pro-triazabutadienes that are generally stablein acidic conditions. For example, carboxylation on N1 yielded apro-triazabutadiene molecule that is stable in concentrated HCl inmethanol; treating the pro-triazabutadiene molecule with NaOH inmethanol returned the original triazabutadiene molecule (which can thenbe degraded, e.g., in acidic conditions). Thus, the pro-triazabutadienemolecule may function as a means to protect triazabutadienes fromdegradation, e.g., under acidic conditions. In some cases the release ofthe triazabutadiene molecule from the pro-triazabutadiene molecule canbe selectively triggered (under appropriate conditions), e.g., viaenzymatic triggers. This can allow for many applications that couldbenefit from selective triazabutadiene activation.

As used herein, the terms “protected triazabutadiene,” “releasabletriazabutadiene” and “pro-triazabutadiene” refer to molecules thatcomprise an inactive form of a triazabutadiene molecule (e.g., aprotected version of a triazabutadiene) but can yield or release anactive triazabutadiene molecule (possibly further yielding an aryldiazonium species) upon appropriate conditions.

The present invention features triazabutadiene molecules, e.g.,water-soluble triazabutadiene molecules. The present invention alsofeatures methods of use (applications) of said triazabutadienemolecules, methods of cleavage of said triazabutadiene molecules (e.g.,decomposition in water, reductive cleavage, pH-dependent cleavage,light-catalyzed cleavage, etc.), and methods of synthesis of saidtriazabutadienes. For example, the present invention features methodsand compositions for yielding an aryl diazonium species from atriazabutadiene molecule, e.g., a protected aryl diazonium species inthe form of a triazabutadiene. In some embodiments, an enzyme catalyzesthe reaction yielding the aryl diazonium species from thetriazabutadiene molecule. The molecules of the present invention (and/orthe products of triazabutadiene molecule cleavage (e.g., diazoniumspecies) may be used for a variety of applications. For example, thepro-triazabutadiene molecules of the present invention may be used indrug delivery systems.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

SUMMARY OF THE INVENTION

The present invention features protected triazabutadienes (orpro-triazabutadienes) that release a triazabutadiene and a derivative ofZ² (see below) when subjected to a trigger that results in deprotectionof the N1 nitrogen. As used herein, the term N1 nitrogen refers to thenitrogen to which Z¹ and Z² are bonded (see below). For example, thepresent invention features compounds of Formula B:

In some embodiments, A is —N—. In some embodiments, D is —H. In someembodiments, E is —H.

In some embodiments, X¹ is an aryl, an alkyl, a carboxylic acid, analcohol, or an amine. For example, in some embodiments, X¹ is methyl. Insome embodiments, X¹ is t-butyl. In some embodiments, X¹ is a moietyconferring water solubility. In some embodiments, X¹ is a moietyconferring a particular pharmacokinetic property. In certainembodiments, X¹ is a moiety of the formula —R¹-Q¹. wherein R¹ is a C₁₋₆alkylene, and Q¹ is a sulfate, phosphate, or a quaternary ammoniumcation.

In some embodiments, Y¹ is an aryl, an alkyl, a carboxylic acid, analcohol, or an amine. In some embodiments, Y¹ is methyl. In someembodiments, Y¹ is t-butyl. In some embodiments, Y¹ is one of thecompounds shown in FIG. 14B. In certain embodiments, Y¹ is a NHS-estermoiety. In certain embodiments, Y¹ is an oligonucleotide. In certainembodiments, Y¹ is a peptide. In certain embodiments, Y¹ is afluorescence quencher. In certain embodiments, Y¹ is a pro-fluorophore.In certain embodiments, Y¹ is an alkyne. In certain embodiments, Y¹ is atriazene. In certain embodiments, Y¹ is a tri-substituted aryl.

In some embodiments, X¹ and Y¹ are both alkyl. In some embodiments, X¹and Y¹ are both methyl. In some embodiments, X¹ and Y¹ are both t-butyl.In certain embodiments, X¹ is a mesityl group. Y¹ is a mesityl group, orboth X¹ and Y¹ are mesityl groups.

In some embodiments, Z¹ is an aryl. In certain embodiments, Z¹ is a drugwith a phenolic functional group. In some embodiments, Z² is a moietycomprising a carbonyl group.

As a non-limiting example, in some embodiments, the compound isaccording to Formula B, wherein A is —N—; D is —H; E is —H; Y¹ is tBu;X¹ is Me; Z1 is aryl, e.g., with amide-linked alkyne or azide; andZ²═COOEt.

In some embodiments, the compound of Formula B is incorporated into anamino acid.

The present invention also features methods of selectively activating atriazabutadiene. The method comprises introducing a trigger to acompound according to Formula B as describe above, wherein the triggerresults in deprotection of N1 nitrogen to cause the compound to yield anactive triazabutadiene molecule and a derivative of Z². In someembodiments, the derivative of Z² is a drug or a pro-drug. In someembodiments, the active triazabutadiene molecule yields an aryldiazonium species.

In some embodiments, the trigger is basic conditions. In someembodiments, the trigger is an enzyme, e.g., a phosphatase, a sulfatase,an esterase, a nitroreductase, etc. In some embodiments, the trigger isa redox environment, e.g., a redox environment inside a cell. In someembodiments, the trigger is light.

In some embodiments, the compound of Formula B is stable in acidicconditions. e.g., a solution having a pH of 8.0 or less, a solutionhaving a pH of 7.0 or less, a solution having a pH of 6.0 or less, etc.

The present invention also features a method of drug release or cargorelease. In certain embodiments, the method comprising introducing anenzymatic trigger to a pro-triazabutadiene molecule according to any theembodiments herein, wherein the enzymatic trigger causes deprotection ofN1 nitrogen thereby causing release of a triazabutadiene molecule and aderivative of Z², wherein the derivative of Z² comprises a drug or acargo. In certain embodiments, the cargo or drug is a pro-drug.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detailed descriptionand claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows non-limiting examples of triazabutadiene molecules.

FIG. 2A shows triazabutadiene molecules undergoing decomposition todiazonium salts (and cyclic guanidine species). Note thereaction/equilibrium arrows are not to scale.

FIG. 2B shows a triazabutadiene molecule breaking down (in low pHconditions) to a diazonium species and a cyclic guanidine species.

FIG. 3 shows reductive cleavage of triazabutadiene molecules.

FIG. 4A shows light catalyzed cleavage of triazabutadiene molecules.

FIG. 4B shows photochemically generated bases. (i) A masked base maydecompose to reveal a basic nitrogen atom upon exposure to light; (ii)The basic nitrogen atom of a molecule obscured by a steric wall may bereversibly swung away in a photochemically triggered fashion; (iii) Theintrinsic basicity of a nitrogen-containing functional group may bealtered by a photochemical event.

FIG. 5A shows time-dependent photo-induced degradation oftriazabutadienes. (i) The reaction was monitored by comparing startingmaterials (A, C, D, and E) with product (B); (ii) Peak absorption andextinction coefficients for all of the compounds were excitable by theUV source used; (iii) Time-dependent conversion of compounds wasmeasured by NMR integration.

FIG. 5B shows (i) Compound A is rendered more basic upon exposure tolight; that basicity recovers (to some extent) in the absence of light;(ii) Oscillating UV irradiation provides a saw-tooth pH trend over time.

FIG. 5C shows the lone-pair of electrons on the N1 nitrogen atom becomesmore electron-rich upon isomerization from E to Z.

FIG. 5D shows the use of the photobase as a catalyst (i) Structures ofwater-soluble Compound A versus organic soluble Compound F; (ii) TheHenry reaction between Compound G and Compound H was carried out at roomtemperature and varying amounts of catalyst; (iii) The reactions weremonitored by ReactIR™, following consumption of aldehyde Compound H.

FIG. 6A-FIG. 6H show non-limiting examples of triazabutadienes orreaction schemes involving triazabutadienes.

FIG. 7A, FIG. 7B, FIG. 7C, and FIG. 7D show non-limiting examples ofreaction schemes involving triazabutadienes.

FIG. 8A shows formulas for releasable triazabutadienes of the presentinvention.

FIG. 88 shows synthesis of a pro-triazabutadiene (Compound 57) from atriazabutadiene (Compound 58) and ethylchloroformate.

FIG. 8C shows a triggered release of a pro-triazabutadiene (Compound58b).

FIG. 8D shows a proposed substrate (a pro-triazabutadiene, Compound 59)for beta-lactamase. Upon cleavage of the beta-lactam, the compound willdecompose to release carbon dioxide and return the triazabutadiene(Compound 58).

FIG. 8E shows synthesis of the pro-triazabutadiene (Compound 59) viaknown beta-lactam (Compound 61).

FIG. 8F shows a tetrahedral intermediate.

FIG. 8G shows synthesis of several protected triazabutadienes that havevarying electronic properties on the aryl ring.

FIG. 8H shows synthesis of a protected triazabutadiene bearing a t-butylester.

FIG. 8I shows removal of the protection of the t-butyl estertriazabutadiene of FIG. 8H under basic conditions.

FIG. 9A shows synthesis of protected triazabutadienes and theirprospective yields using various chloroformate reagents.

FIG. 9B shows a hydrolysis reaction of protected triazabutadienes whereR═—CH₂CH₃ in buffer. The reaction is assumed to be second order with theaddition of hydroxide to be the rate-determining step.

FIG. 9C shows a decrease in absorbance of the protected triazabutadienewhere R═—CH₂CH₃ as the hydrolysis reaction proceeds over time. Compoundwas in a concentration of 31 μM in 25 mM sodium borate buffer of pH 10with scans every 60 seconds.

FIG. 9D shows the concentration (μM) of protected triazabutadiene whereR═—CH₂CH₃ vs. time (min). Scans were obtained every 60 seconds. Initialconcentration of compound was 31 μM in buffers of various pH ranges.Buffers pH 8, 9, and 10:25 mM sodium borate; buffer pH 7:100 mMphosphate; buffer pH 2:200 mM KCl buffer.

FIG. 9E shows a graph of ln(abs) vs. time (s) to obtain K_(obs) underpseudo-first order reaction conditions: a plot of K_(obs) vs. hydroxideconcentration (M) to obtain the second order rate constant (k₂). Thechart shows various protected triazabutadienes with their correspondingsecond order rate constants (k₃).

FIG. 9F shows synthesis of a protected triazabutadiene with aphotocleavable protecting group.

FIG. 9G shows a photocleavable protected triazabutadiene exposed to 365nm light to yield a regular triazabutadiene.

FIG. 9H shows a non-limiting example of a triazabutadiene that isphotocleavable but not cleavable using acids or bases.

FIG. 10A shows an example of a triazabutadiene molecule adapted tomodify a protein.

FIG. 10B shows an example of a triazabutadiene molecule conjugated to anantibody, wherein the conjugate is used for labeling a protein ofinterest.

FIG. 11A shows synthesis of a triazabutadiene containing anN-hydroxysuccinimide ester.

FIG. 11B shows Compound 68.

FIG. 11C shows derivatives related to Compound 68.

FIG. 12 shows lysine-reactive triazabutadiene Compound 80 is designed toself-immolate to return the starting lysine residue if the resultingdiazonium ion, Compound 81, fails to undergo a reaction with tyrosine.

FIG. 13A shows an example of cargo release from a triazabutadienemolecule.

FIG. 13B shows an example of how a prodrug is released.

FIG. 13C shows an example of a prodrug comprising a phenolic functionalgroup masked as a triazylidine moiety.

FIG. 13D shows an azide functional group reacted with a carbine toproduce an acid labile prodrug comprising a triazylidine moiety.

FIG. 14A shows an example of triazabutadiene (left molecule) and areaction to create a protected the triazabutadiene (middle molecule),e.g., a triazabutadiene with N1 nitrogen protection to help prevent therapid release of the aryl diazonium that may typically occur if thetriazabutadiene is not protected. The protected triazabutadiene may,upon an enzymatic trigger (right molecule), yield an aryl diazoniumspecies.

FIG. 14B shows Formula B (left) and non-limiting examples of Y¹.

DETAILED DESCRIPTION OF THE INVENTION I. Triazabutadiene Molecules

The present invention features triazabutadiene molecules (e.g.,water-soluble triazabutadienes). Non-limiting examples of formulas fortriazabutadiene molecules of the present invention are of shown inFIG. 1. For example, in some embodiments, triazabutadienes are accordingto Formula A. Examples of Formula A are shown as Formula I, II, III, andIV. The present invention is not limited to Formula A, Formula I,Formula II, Formula III, and Formula IV. Referring to FIG. 1, in someembodiments, A=S, O, or N. (Note in some embodiments, if A=S, Y¹ and X¹may be switched.) In some embodiments, D=H, —CH═CH—CH=E-, halides,cyano, sulfonates, alkyl chain, or trifluoromethyl. In some embodiments,E=H, —CH═CH—CH=D-, halides, cyano, sulfonates, alkyl chain, ortrifluoromethyl.

In some embodiments, X¹ is a moiety conferring water solubility. Incertain embodiments, Y¹ is a tri-substituted aryl, an alkyl, acarboxylic acid, an alcohol, or an amine (see also FIG. 14B). In someembodiments, Y¹ is a tri-substituted aryl group, the tri-substitutedaryl group comprising a NHS-ester moiety (e.g., for protein linkage); anoligonucleotide; a peptide; a fluorescence quencher; a pro-fluorophore;an alkyne (e.g., for click chemistry); a triazene (e.g., from clickreaction); the like, or a combination thereof. In some embodiments, Y¹comprises an aldehyde; an amine (e.g., Fmoc protected), aminooxy,halogen (e.g., radio isotope); the like, or a combination thereof. Insome embodiments, Z¹ is an aryl, e.g., a substituted aryl. In someembodiments, Z¹ comprises a NHS-ester moiety; an oligonucleotide; apeptide; a fluorescence quencher; a pro-fluorophore; a biologicallyactive acid labile compound; a prodrug comprising a phenolic functionalgroup; releasable cargo; an alkyne (e.g., for click chemistry); atriazene (e.g., from click reaction); the like, or a combinationthereof. In some embodiments, Z¹ comprises an aldehyde; an amine (e.g.,Fmoc protected), aminooxy, halogen (e.g., radio isotope); the like, or acombination thereof.

In some embodiments, X¹ may comprise a functional group that conferswater solubility. In some embodiments, X¹ comprise a moiety of theformula —R¹-Q¹, wherein R¹ is C₁-6 alkylene, and Q¹ is sulfate,phosphate, or a quaternary ammonium cation. In some embodiments, X¹ is amoiety of the formula —R¹-Q¹, wherein R¹ is C₁₋₆ alkylene, and Q¹ issulfate (e.g., —(O)_(n)SO₃R^(a), where n is 0 or 1, and R^(a) is C1-6alkyl or typically H), phosphate (e.g., —(O)_(n)PO₃R^(a), where n is 0or 1, and R^(a) is C1-6 alkyl or typically H), or a quaternary ammoniumcation (e.g., —[NR^(a)R^(b)R^(c)]⁺, where each of R^(a), R^(b), andR^(c) is independently H or C₁₋₆ alkyl). As used herein, the term“alkyl” refers to a saturated linear monovalent hydrocarbon moiety ofone to twelve, typically one to six, carbon atoms or a saturatedbranched monovalent hydrocarbon moiety of three to twelve, typicallythree to six, carbon atoms. Examples of alkyl groups include, but arenot limited to, methyl, ethyl, n-propyl, 2-propyl, tert-butyl, pentyl,and the like. The term “alkene” refers to a saturated linear divalenthydrocarbon moiety of one to twelve, typically one to six, carbon atomsor a branched saturated divalent hydrocarbon moiety of three to twelve,typically three to six, carbon atoms. Examples of alkene groups include,but are not limited to, methylene, ethylene, propylene, butylene,pentylene, and the like.

Triazabutadiene molecules of the present invention may be readilysoluble in water. In some embodiments, the solubility of thetriazabutadiene molecule in water is at least 23 g/L of water (50 mM).In some embodiments, the triazabutadiene molecule is stable in pH 7.4phosphate buffer. The phosphate buffer solutions are commerciallyavailable or can be prepared, for example, as described inhttp://cshprotocols.cshlp.org/content/2006/1/pdb.rec8247. In someinstances, the half-life of the triazabutadiene molecules of the presentinvention in pH 7.4 phosphate buffer solution is at least 24 hours.

Stability of the triazabutadiene molecule may be measured in variousways. In some embodiments, stability is measured by the half-life of themolecule (or the half-life of the molecule in a particular buffer at aparticular pH). In some embodiments, the molecule has a half-life of atleast 12 hours in a pH 7.4 buffer. In some embodiments, the molecule hashalf-life of at least 24 hours in a pH 7.4 buffer. In some embodiments,the molecule has half-life of at least 36 hours in a pH 7.4 buffer. Insome embodiments, the triazabutadiene molecule has a half-life of atleast 8 hours, at least 10 hours, at least 12 hours, at least 20 hours,at least 24 hours, at least 30 hours, at least 36 hours, etc. Thepresent invention is not limited to the aforementioned examples ofstability measurements.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the triazabutadiene molecules of thepresent invention are advantageous because the triazabutadiene moleculescan be easily modified (e.g., various different functional groups can beeasily used as X¹, Y¹, or Z¹ (see FIG. 1). And, the release of thediazonium species following triazabutadiene molecule breakdown (viacertain mechanisms, as described below) may provide a new functionalgroup that can be taken advantage of in various applications. Also, itmay be considered advantageous that the breakdown of the triazabutadienemolecule is irreversible.

II. Cleavage of Triazabutadiene Molecules

a. Water and/or Low pH

The present invention shows that triazabutadiene molecules may breakdown in the presence of water to generate reactive aryl diazoniumcompounds. For example, FIG. 2A shows that triazabutadiene molecules ofthe present invention can undergo decomposition to diazonium salts(reactive aryl diazonium compounds) and cyclic guanidine species. Aryldiazonium compounds may react with electron-rich aryl rings (e.g., arylspecies wherein the bond of interest is a nitrogen-carbon bond; indoles,anilines, phenol-containing compounds such as resorcinol or tyrosine,etc.) to form stable azobenzene linkages (e.g., an aryl azo dye, e.g.,Sudan Orange). (Note the present invention is not limited to theaforementioned phenol-containing species. In some embodiments, imidazolecompounds (e.g., purine bases like guanine) may be used in lieu of aphenol-containing compound.) The diazonium species may not necessarilyreact with an electron-rich aryl rings compound (e.g., phenol species),for example if a phenol species is not present. The diazonium speciesmay irreversibly extrude nitrogen gas to generate an aryl cation, whichwill rapidly be quenched by solvating water, thus synthesizing a newphenolic compound (e.g., HO-Ph, wherein Ph refers to the phenyl ring);thus, the diazonium portion of the triazabutadiene molecule may functionas a masked hydroxyl group.

In some embodiments, the triazabutadiene molecules are acid labile,e.g., unstable at particular pH levels (see FIG. 2B). For example,decreases in pH may increase the rate at which the triazabutadienemolecules break down (the half life of the molecule decreases). In someembodiments, the triazabutadiene molecules are unstable at low (lowered)pH levels (e.g., lowered pH as compared to a particular pH that themolecule may be stored at, e.g., a pH wherein the molecule has aparticular desired half life). Low pH levels, in some example, may be asub-physiological pH (7.4 or less). In some embodiments, thetriazabutadiene molecules are (more) unstable at pH 7.0 or less, pH 6.8or less, pH 6.5 or less, pH 6.2 or less, pH 6.0 or less. pH 5.8 or less,pH 5.6 or less, pH 5.5 or less, pH 5.2 or less, pH 5.0 or less, etc.

The term ‘low pH” may refer to several different pH levels. Since thefunctional groups attached to the molecule (e.g., see X¹, Y¹, Z¹ ofFormula I) affect the stability of the molecule (as well as watersolubility), the pH that is necessary to increase the rate of breakdownof the triazabutadiene molecule (e.g., the “lowered pH”) may bedifferent for different molecules. In some embodiments, the low pH is apH of 7.4 or less, 7.2 or less, 7.0 or less, etc. In some embodiments,the low pH is a pH of 6.8 or less, 6.6 or less, 6.5 or less, 6.4 orless, 6.2 or less, 6.0 or less, etc. In some embodiments, the low pH isa pH of 5.8, 5.5 or less, 5.0 or less, etc.

In some embodiments, the triazabutadiene molecules can break downwithout the presence of the low pH (the molecules have half lives);however, in some embodiments, a lowered pH enhances the reaction (e.g.,increases the rate of reaction). As such, a low pH may or may not beused with the molecules and/or methods of the present invention. In someembodiments, the triazabutadiene molecule has a half-life of no morethan 1 hour in a pH 7.4 aqueous solution. In some embodiments, thetriazabutadiene molecule has a half-life of no more than 30 minutes in apH 7.4 aqueous solution. In some embodiments, the triazabutadienemolecule has a half-life of no more than 15 minutes in a pH 7.4 aqueoussolution.

The present invention also features methods of breaking downtriazabutadiene molecules. In some embodiments, the method comprisessubjecting the molecule to water. In some embodiments, the methodcomprises subjecting the molecule to a low pH (e.g., a low pH that isappropriate for the molecule, e.g., a lowered pH that increases the rateat which the triazabutadiene molecule breaks down).

In some embodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 10-30 seconds. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 30 seconds to 1 minute. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 1 to 5 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 5-10 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 10-15 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 15-20 minutes. In someembodiments, the reaction of the triazabutadiene molecule to thediazonium species occurs in water within 25 minutes, within 30 minutes,within 45 minutes, or within 60 minutes.

In some embodiments, the diazonium species may be visuallydifferentiated from the triazabutadiene species, e.g., the diazoniumspecies is visually distinct (e.g., a different color) from thetriazabutadiene molecule. If applicable, in some embodiments, the arylazo dye may be visually differentiated from the triazabutadiene speciesand the diazonium species, e.g., the aryl azo dye is visually distinct(e.g., a different color) from the triazabutadiene species and thediazonium species.

Given the possibility that the aryl azo dye is visually distinct fromthe triazabutadiene molecule (and/or the diazonium species), the presentinvention also features methods of producing a visually detectablemolecule. In some embodiments, the method comprises providing atriazabutadiene molecule according to the present invention andsubjecting the triazabutadiene molecule to water and/or a low pH (orlight as discussed below, or light and low pH, etc.). The low pH (orlight, or light and low pH, etc.) initiates (e.g., increases the rateof) the irreversible reaction to produce the diazonium species and thecyclic guanidine species. As previously discussed, the diazonium speciesmay be visually distinct from the triazabutadiene molecule; thereforethe reaction produces a visually detectable molecule.

b. Reductive Cleavage

Other mechanisms may be used to break down triazabutadiene molecules ofthe present invention. For example, in some embodiments, reducingconditions increase the rate at which the triazabutadiene moleculesbreak down. Thus, the present invention also features methods ofreductive cleavage of triazabutadiene molecules. For example,triazabutadiene molecules (e.g., triazabutadiene scaffolds) may bereadily cleaved using reducing agents such as but not limited to sodiumdithionite (sodium hydrosulfite) (Na₂S₂O₄) (see FIG. 3). In someembodiments, the reducing agent comprises lithium aluminum hydride,sodium borohydride, or the like.

In some embodiments, electrochemical reduction may be used in accordancewith the present invention. Reductive cleavage of the triazabutadienemolecules provides a urea functionality and a terminal aryl triazene. Insome embodiments, the aryl triazene is further reduced in the presenceof excess reducing agent (e.g., sodium dithionite). In some embodiments,the reduction can be observed visually by the change in color of asolution. For example, there may be a subtle change of yellows thatresults from a loss of a shoulder in UV/vis spectrum.

In some embodiments, the ratio of the concentration of thetriazabutadiene to the reducing agent is about 1:1. In some embodiments,the ratio of the concentration of the triazabutadiene to the reducingagent is about 1:2. The present invention is not limited to theaforementioned ratios. For example, in some embodiments, the ratio ofthe concentration of the triazabutadiene to the reducing agent is about2:3, 4:5, etc. The present invention is not limited to theaforementioned ratio of concentrations.

In some embodiments, the reduction can occur within about 10 minutes,within about 15 minutes, within about 20 minutes, within about 25 min,within about 30 min, etc., at room temperature. Without wishing to limitthe present invention to any theory or mechanism, it is believed thatreductive cleavage of the triazabutadiene molecules is advantageousbecause it can occur rapidly (e.g., within 10 minutes, within 15minutes). Also, the triazabutadiene molecules that are highly stable inacid (e.g., a p-CN derived triazabutadiene) may still be susceptible toreducing conditions.

In some embodiments, reductive cleavage of triazabutadiene molecules mayalso be used to cleave unreacted triazabutadienes that did not undergodiazonium formation/reaction chemistry that is associated with a drop inpH (or other mechanism) as described above (a sort of quench for the pHchemistry).

c. Light-Initiated Cleavage

In some embodiments, light increases the rate at which thetriazabutadiene molecule breaks down (into the cyclic guanidine speciesand the diazonium species) (see FIG. 4A). The present invention alsofeatures water-soluble triazabutadienes that, upon photo-irradiation,may be rendered more basic in a reversible fashion. Referring to FIG.4B, for reference, a protecting group of a masked base may decompose toreveal a basic nitrogen atom upon exposure to light. Or, a basicnitrogen atom of a molecule obscured by a steric wall may be reversiblyswung away in a photochemically-triggered manner. The present inventionshows the intrinsic basicity of a nitrogen-containing functional groupmay be altered by a photochemical event.

Referring to FIG. 5A, in some embodiments, triazabutadiene molecules ofthe present invention may readily photoisomerize to a more reactiveZ-form. As an example, an aqueous solution of Compound A was irradiatedwith a simple hand-held UV lamp (“365 nm,” measured at 350 nm).Consumption of Compound A was observed after only a few hours. Thenon-irradiated reaction under similar conditions was stable for days aspartial degradation rapidly rendered the solution mildly basic. Withoutwishing to limit the present invention to any theory or mechanism, itwas hypothesized that if a two-electron process were happening, thenCompound A-Z would be more basic than Compound A-E. A 1.0 N NaOHsolution of Compound A was treated with light. At pH 14, Compound A wasstable for weeks in the dark; it was surprisingly discovered that nearcomplete consumption of starting material after 20 hours of constantirradiation occurred. NMR analysis of samples post-irradiation showedcyclic guanidine Compound B. Evidence of a benzene diazonium species orphenol/azobenzene products derived therefrom was not observed. Benzenediazonium ions also absorb UV light to expel nitrogen and generate abenzene radical. In order to resolve if the initial cleavage undergoes aradical homolytic mechanism versus a two-electron heterolytic mechanism,a trapping experiment using resorcinol was conducted. (Resorcinol waschosen because it can serve a dual role as a radical scavenger and atrap benzene diazonium species that could be formed.) An excess ofresorcinol was added to a pH 9 borate-buffered solution of Compound Aand the mixture was irradiated with light. The known azobenzene, SudanOrange G, was formed in a 65% yield (versus 4% for the non-irradiatedreaction). Derivatives of Compound A were made to examine the effects ofelectronic perturbations on the light-induced degradation. Electrondeficient aryl rings are more stable at lower pH, and this trendgenerally holds true for the photochemical reactions as well. A bufferedborate solution was chosen due to its alkaline nature and lack ofcomplicating signals in the NMR experiment. Compounds C-E all haveabsorption spectra that are well within the range of the UV lamp (seeFIG. 5A(ii)). Both m-NO₂ (Compound C) and p-CN (Compound D) had similarrates of reaction, both slower than Compound A. To rule out othereffects associated with possible heating or interactions of the buffer,p-NO₂ derivative Compound E was irradiated because of its significantlyred-shifted spectrum. Compound E absorbed in a range that was notirradiated with the UV lamp and as such was recalcitrant to degradation(see FIG. 5A(iii)).

As previously discussed, poorly (or non-) buffered aqueous solutionscould become more basic as a function of time due to the degradation toCompound B and the aryl diazonium species. Without wishing to limit thepresent invention to any theory or mechanism, it is believed that thecause of the increase in pH is Compound B, which acts as a base. It wasfound that reactions slowed and eventually stopped once the pH had risento around 9. Without wishing to limit the present invention to anytheory or mechanism, it was hypothesized that by driving the reaction tocompletion with light, it would be possible to increase the pH beyondthis dark-reaction imposed wall (analogous FIG. 4B(ii)). Using NMR and apH meter, it was observed that the pH of a solution of Compound Airradiated with UV light rose in a time-dependent manner.

In an effort to examine the rate order for the pH-increasing reactionmore carefully, in situ, real-time pH measurements were acquired.Compound A was dissolved in water and the pH of the solution wasadjusted to 9 such that it would not form Compound B in the absence oflight. Upon exposing the solution to 350 nm light, it was surprisinglydiscovered that the solution rapidly spiked up to a pH of ˜10 over thecourse of several minutes, and only upon much longer exposure slowlybecame more basic. This spike was not at all consistent with the modelof the pH increase being solely linked to the concentration of CompoundB being generated. Moreover, previous NMR studies showed that much moretime was required to afford a pH change commensurate with this apparentlevel of degradation.

Without wishing to limit the present invention to any theory ormechanism, it was hypothesized that the rapid pH increase that wasobserved was not attributed to Compound B, but instead a result of the Zisomer being significantly more basic than the E isomer (see FIG.5B(i)). A sample was irradiated and then the light was turned off oncethe pH of the solution started to increase noticeably. As the samplethermally reverted to the more stable E form, the pH of the solutiondropped as well (see FIG. 5B(ii)). The experiment was repeated withincreasing times of irradiation, and a saw-tooth pattern was obtained.The process was not completely reversible due to some degradation toCompound B. Indeed, triazabutadiene Compound A can serve a dual role ofbeing a photo-masked base (see FIG. 4B(i)), and a base whose intrinsicfunctional group properties are altered photochemically (FIG. 4B(iii)).

This phenomenon via an isomerization-induced pK_(b) change wassurprisingly discovered by the inventor. Without wishing to limit thepresent invention to any theory or mechanism, unlike the case whereHecht's compound is rendered basic upon irradiation by way of moving ofa steric wall (see FIG. 4B(ii)), it may be unlikely that steric factorsplay a significant role in this chemistry, e.g., in water. It ispossible that the E isomer has alternating non-π involved lone pairs ofelectrons, whereas the Z isomer has two adjacent lone pairs of electrons(see FIG. 5C). The electronic repulsion from these renders N1 much moreelectron rich, and thus a stronger Lewis base.

Referring to FIG. 5A(iii), Compound C and Compound D were examined in aneffort to find a base that was reversibly basic but also more resistantto degradation. In both cases, a slow subtle change to the pH wasobserved, but none as dramatic and rapid as Compound A. Without wishingto limit the present invention to any theory or mechanism, it isbelieved that this may be due to factors such as (a) faster thermalisomerization to the E isomer such that a build up of the Z isomer isnot possible; (b) the electron-deficient triazabutadienes are less basicto begin with, so a transition is not observable in the operating pHrange.

It is possible that Compound A may be useful as a photo-catalytic basein the context of organic reactions. With limited solubility in all butDMSO, the stability of Compound A was tested. As noted previously,Compound A is quite stable to an excess of acetic acid in DMSO, showingonly 12% degradation over 14 hours at room temperature. Upon irradiationwith light, Compound A in presence of acetic acid completely fell apartover the same time frame. To confirm that this was due to the acid, asolution of Compound A (in pure DMSO) was irradiated. After four hoursof constant irradiation in acid-free DMSO, an E:Z ratio of nearly 50:50was observed. Moreover, unlike in water, the thermal reversion from Z toE is slow in pure DMSO with a half-life on the order of days.Attributing this to lack of protonation, a control in MeOD was run, anda first-order thermal isomerization was observed with a rate of 3×10⁻⁵s⁻¹ (t_(1/2)˜6.4 hours), in addition to some degradation to Compound B.

Referring to FIG. 5D, due to the limited organic solubility of CompoundA, Compound F (FIG. 5D(i)) was synthesized. With Compound F, a similarlight-induced acid sensitivity was observed in DMSO (and slow thermalisomerization). Based on the apparent pK_(b) of Compound F, pK_(a) werematched to condensation substrates. A Henry reaction between nitroethane(Compound G) and p-nitrobenzaldehyde (H) was chosen to demonstrate thevirtues of Compound F (FIG. 5D(ii)). The reaction between Compound G andCompound H occurred rapidly at room temperature in a light and catalystdependent manner (FIG. 5D(iii)). The reaction with 25 mole % Compound Fin the absence of light was exceedingly slow. Likewise, the reactionwith light but no catalyst also failed to proceed. The cyclic guanidinewas not observed during a post-reaction analysis of the components froma 25 mole % Compound F run, indicating that the Z-isomer of Compound Fis likely to be the catalytically active species in solution. Slowthermal isomerization back to the E-isomer in aprotic organic solventstogether with a fast overall reaction attempts to adjust the reactionrate prior to consumption of Compound H. Interestingly, the reactioncatalyzed with Compound F was significantly faster than the samereaction reported by Hecht. This may provide evidence that Compound F-Zis more basic than Hecht's blocked trialkylamine.

As previously discussed, the present invention features methods ofbreaking down triazabutadiene molecules by subjecting the molecule tolight. The light may, for example, include wavelengths of about 400 nm.The present invention is not limited to wavelengths of 400 nm or about400 nm. For example, in some embodiments, the wavelength is from 350 nmto 400 nm (e.g., 370 nm). In some embodiments, the wavelength is from360 nm to 410 nm. In some embodiments, the wavelength is from 330 nm to420 nm. In some embodiments, the wavelength is from 340 nm to 430 nm. Insome embodiments, the method comprises subjecting the molecule to a lowpH and to light.

As previously discussed, light-promoted reactivity andlight-facilitating E/Z isomerization has been observed. In someembodiments, a system such as a UV-LED pen may be used for thesereactions, however the present invention is not limited to a UV-LED penand may utilize any appropriate system. The UV-LED pens may allow forrelatively narrow bandwidth irradiation of these compounds (but are notlimited to these bandwidths). The color of the bulk material shifts as aresult of electronic perturbations to the aryl azide starting material.For example, nitro derivative Compound 6e of FIG. 6G (described below)is rust-red, versus an orange phenyl Compound 6c of FIG. 6F (describedbelow) and yellow-orange methoxy Compound 6d of FIG. 6G (describedbelow). It may be possible for selective irradiation of a complexmixture in an orthogonal sense. These experiments may be performed inbasic aqueous solutions to maintain the solvation properties of waterwhile also preventing the degradation pathway stemming from protonation.These experiments are not limited to basic aqueous solutions.

Without wishing to limit the present invention to any theory ormechanism, it may be considered advantageous that the breakdown of thetriazabutadiene molecule is irreversible.

III. Synthesis of Water-Soluble Triazabutadiene Molecules andExperimental Examples

Synthesis of 1-mesityl-1-H-imidazole: To a solution of2,4,6-trimethylaniline (1.35 g, 10.0 mmol) in methanol (15 mL) □wasadded a solution of glyoxal (40%) (1.14 mL, 40% in water, 10. mmol). Themixture was stirred at room temperature until a solid formed.Thereafter, solid ammonium chloride (1.07 g, 20 mmol), formaldehyde(37%) (1.6 mL 37% in water, 60. mmol) and methanol (40 mL) were added,and the mixture was heated to reflux for one hour. After the hour,phosphoric acid (1.4 ml of an 85% solution) was added drop wise and themixture was refluxed for an additional eight hours. Upon cooling to roomtemperature ice (30 g) was added and the solution was brought to a pH of9 with potassium hydroxide (40% in water). The following mixture wasextracted repeatedly with diethyl ether. The ether phase was dried overmagnesium sulfate and solvent removed in vacuo to form a brown solidwhich was filtered and washed with hexanes to give the product (0.785 g;42%). 1H NMR (500 MHz, CDCl3): δ 7.45 (t, J=1.1 Hz, 1H), 7.25 (t, J=1.1Hz, 1H), 6.99 (dp, J=1.3, 0.7 Hz, 2H), 6.91 (t, J=1.3 Hz, 1H), 2.36 (t,J=0.7 Hz, 3H), 2.01 (t, J=0.6 Hz, 6H). 13C NMR (126 MHz, CDCl3) δ138.80, 137.47, 135.42, 133.40, 129.55, 128.96, 120.02, 21.03, 17.33(see Liu, J. et al. Synthesis 2003, 17, 2661-2666).

Synthesis of 3-(1-mesityl-1H-imidazol-3-ium-3-yl) propane-1-sulfonate(e.g., see FIG. 5A): To a solution of 1-mesityl-1-H-imidazole (1.00 g,5.36 mmol) in toluene (30 mL) was added 1,3-propanesultone (1.00 g, 8.18mmol) and the mixture was heated to reflux overnight. The mixture wasallowed to cool to room temperature and the off-white precipitatecollected by filtration. The precipitate was further washed with diethylether and dried using a vacuum oven to yield a solid (1.40 g; 84%). 1HNMR (500 MHz, D2O): δ 8.92 (t, J=1.6 Hz, 1H), 7.75 (t, J=1.8 Hz, 1H).7.49 (t, J=1.8 Hz, 1H). 7.06 (q, J=0.8 Hz, 2H), 4.44 (t, J=7.1 Hz, 2H),2.39-2.31 (m, 2H), 2.25 (s, 3H), 1.96 (s, 6H). 13C NMR (126 MHz, D2O) δ141.42, 136.54, 134.64, 130.74, 124.34, 123.00, 48.18, 47.17, 25.03,20.17, 16.29.

Synthesis of Potassium 3-(3-mesityl-2-(phenyltriaz-2-en-1-ylidene)-2,3-dihydro-1H-imidazol-1-yl) propane-1-sulfonate (e.g., see FIG. 6B): Toa slurry of 3-(1-mesityl-1H-imidazol-3-ium-3-yl)propane-1-sulfonate (50mg, 0.16 mmol) in dry THF (6 mL), was added a solution of phenyl azidein THF (0.16 mL, 1 M, 0.16 mmol). To the solution was added KO-t-Bu (24mg, 0.21 mmol) in one portion and the resulting mixture was stirredunder argon for 4 hours. Hexanes (1 mL) was then added and the reactionmixture was filtered. The solvent was removed and the residue taken upin a minimal amount of DCM and on trituration with hexanes, pure productwas obtained by filtration as a yellow powder (61 mg, 81%). 1H NMR (500MHz, DMSO-d6) δ 7.32 (d, J=2.4 Hz, 1H), 7.07-7.02 (m, 4H), 6.99-6.94 (m,1H), 6.84 (d, J=2.4 Hz, 1H), 6.51-6.47 (m, 2H), 4.09 (t, J=7.1 Hz, 2H),2.34 (s, 3H), 2.12-2.04 (m, 2H), 1.95 (s, 6H). 13C NMR (126 MHz,DMSO-d6) δ 152.19, 151.13, 137.94, 136.15, 134.31, 129.31, 128.60,125.26, 120.90, 117.61, 117.24, 48.52, 45.05, 25.80, 21.06, 17.95.

Using the procedures described herein, the p-methoxy and p-nitro analogs(from the p-MeO aryl azide and p-NO2 aryl azide) were also prepared.

For decomposition experiments, buffers were made to the appropriate pHin a 9:1 mix of H2O:D2O. These solutions were added to the compoundbeing assayed such that the buffer capacity was at least 10 fold theconcentration of the compound. Some experiments used 5 mg compound in0.5 mL of buffer (e.g., pH 5 acetate buffer at 25 degrees C.). Thesewere immediately inserted into an NMR instrument and scans were taken ateven time intervals to calculate the half-life of the compound based onintegration.

As another non-limiting example, an azide (e.g., NHS-azide) toN-heterocyclic carbene (NHC) route may be used to synthesizetriazabutadiene molecules (e.g., see FIG. 6C). For example, as shown inCompound 4 of FIG. 6D, a triazabutadiene molecule was synthesized fromdimethyl imidazole derived NHC and phenyl azide. When thetriazabutadiene molecule (Compound 4) was treated with methanolic HCL, arapid color change occurred. This change was confirmed to coincide withdiazonium formation by trapping the reactive species with resorcinol toprovide known diazo dye Sudan Orange G. When the triazabutadienemolecule (Compound 4) was treated with the much less acidic acetic acid,the same product was obtained. Compound 4 was not water-soluble.

To render the triazabutadiene water-soluble, methyl imidazole wasalkylated with propane sultone to provide the Zwitterionic NHC precursorCompound 5a (see FIG. 6E). Formation of the NHC under basic conditionsin the presence of phenyl azide yielded the highly water solubleCompound 6a (see FIG. 6E). Compound 6a was highly colored, so its pHdependence was studied using UV/Vis. The reactions were not only pH-,but also scan-frequency dependent. Upon finding this, the stability ofCompound 6a was studied in D2O in the dark using NMR. Even in the darkit was unstable, but not in the diazonium-forming way. Both Compound 6band a more hindered mesityl (Mes) substituted Compound 6c (see FIG. 6E)were synthesized to stabilize what was initially considered to be arearrangement pathway that could be blocked by steric repulsion.Compound 6c was the most stable of the three (less than 10% consumedafter 24 hours versus 50% for Compound 6a and Compound 6b). Dissolutionin 0.1 N NaOH rendered all compounds stable (e.g., no detectabledegradation after 24 hours in the dark).

As mentioned above, Compound 6c was reasonably stable in pure D2O. Uponadjusting the pH to 5 with HCl, a rapid initial consumption of Compound6c to Compound 7 (see FIG. 6E) and a benzenediazonium salt was noted.After this initial burst of reactivity, a slowing and apparent arrestingof the reaction was noted. At this pH the hydronium was the limitingreagent. All future reactions were run in buffers with a buffer capacitysufficient to maintain a large excess of hydronium ions. The experimentswere performed in 90:10 H2O:D2O buffered solutions to minimizeconsiderations of pH vs. pD. The decomposition to diazonium salts andCompound 7 was measured as a function of pH in phosphate/citrate buffersfrom pH 4-7 and in a phosphate buffer from pH 6-8. All runs providedlinear correlations of concentration and time, indicating a pseudo-zeroorder reaction (first order with respect to hydronium ion with a largeexcess of hydronium ions). While the peaks for Compound 7 remainedconstant, the peaks associated with Compound 6c drifted downfield as thereaction progressed. This drifting was highly reproducible acrosssamples and buffers, but the underlying cause is not understood at thistime. A sigmoidal correlation between rate and buffer pH centered at pH6 was obtained. When resorcinol was not added to consume the diazoniumspecies, 4-phenylazophenol (Compound 8) was observed (see FIG. 6F).Compound 8 came from the decomposition of one diazonium ion to phenolfollowed by reaction with a second diazonium ion. The instability ofCompound 6c in a pH 7 phosphate buffer was surprising given thestability in D2O. Compound 6c was tested in a non-buffered 90:10 H2O:D2Osolution and observed only >7% after 6 hours.

To further examine the reactivity of this class of compounds, variantsCompound 6d and Compound 6e were synthesized (see FIG. 6G). It washypothesized that the p-methoxy and p-nitro analogs (Compound 6d andCompound 6e, respectively) would display different reactivity profiles.It was observed that in pure D20, 26% of Compound 6d was consumed after24 hours in the dark at room temperature as compared with Compound 6e,which was stable to within the detection limit of NMR. Preliminary datashows that Compound 6d undergoes decomposition to the diazonium speciesmore rapidly than Compound 6c in pH 5, 6, and 7 phosphate/citrate buffer(rates of 2.0×10-5, 1.0×10-5, and 0.53×10-5 M/s, respectively). Uponattempting the same study with Compound 6e it rapidly precipitated outof solution across the same pH range. After collecting the precipitateand dissolving it in deuterated methanol, no change was observed from asample of Compound 6e that had never been exposed to a bufferedsolution. Treatment of this methanolic Compound 6e with HCl led to animmediate color change and diazonium formation was confirmed by trappingwith resorcinol. It is possible that 1) that the sodium salt of Compound6e is much less soluble than the potassium salt; or 2) with differentsolvating ions present the sulfonate interacts with the electron-poor N2nitrogen atom of the triazabutadiene to break conjugation and form aninsoluble complex (this is backed by a reversible color change of thestarting rust-red solid, to the light yellow precipitate). Note that thep-nitrobenzenediazonium salts are reported to have the best labelingefficiency of tyrosine residues on proteins.

The influence of solvated ions on reactivity was studied. In water, or aheavy water/water mixture, a near-zero rate of diazonium salt formationwas observed, yet in solutions buffered to pH 7 and even pH 7.4, anincrease in the reaction rate was observed. To assess the role of theanionic component, the reaction in the presence of a range of bufferswhile holding the pH constant may be observed. Buffers include but arenot limited to those expected to have the most diverse properties, e.g.,MES, a Zwitterionic morpholino sulfonic acid, and imidazolium chloride,the conjugate acid of a mild base, can both buffer a solution at pH 6.5,but ionic species in solution would be dramatically different. Themetals in solution could well be acting as Lewis acids to activate ourmolecule. A range of metal halide salts dissolved in pure water atvarying concentrations may be screened.

Note that all of the compounds in the 6 series (FIG. 6E, FIG. 6F, andFIG. 6G) have a built-in sulfonate to confer solubility. It is possiblethat this functional group could be serving an important role byeffecting the localization of metals, directing them to interact withthe nitrogen atoms of the triazabutadiene and thus alter the reactivityof the compound. This may be happening with Compound 6e to such anextreme that the compound is no longer soluble. This concept of adirected metal binding on triazabutadienes was observed, albeit in anorganic environment. Referring to FIG. 6H, to study the role of the sidechain, the imidazole core will be alkylated (Compound 9) with eitherbutane sultone to provide imidazolium (Compound 10) and triazabutadiene(Compound 11), or a dialkyl aziridinium salt to provide the analogousCompound 12 and Compound 13 which invert the expected charge on theside-chain. The extra methylene in Compound 11 as compared with Compound6 may alter the way that the side-chain bites back on thetriazabutadiene. The tertiary amine will be protonated at physiologicalpH and as serve to invert the charge of the side arm. Without wishing tolimit the present invention to any theory or mechanism, a potentialbonus of Compound 13 is that the basic nitrogen may help localize thiscompound in the most acidic subcellular compartments much likeLysoTracker™ dyes.

Regarding the role of mesityl group in reactivity, it is possible that afunction of the mesityl in triazabutadiene reactivity is to provide asteric wall to prevent side reactions. The NMR of Compound 6c (see FIG.6F) shows a tale of two hydrogen atoms on the imidazole ring. Withoutwishing to limit the present invention to any theory or mechanism, it isbelieved that because the ortho methyl groups prevent coplanar arylrings, the mesityl group is unlikely to sit in conjugation with theimidazole, but the highly differentiated chemical environments might beexplained by: 1) the mesityl Tr-system deshielding the adjacent hydrogenatom, and 2) the aryl ring having an inductive effect. Changing thep-methyl of the mesityl to electron donating and withdrawing groups mayallow the adjustment of the electronic parameters without disrupting thesteric bulk.

Referring to FIG. 7A, in some embodiments, synthesis may be performedwith known p-azido dimethyl aniline (Compound 14) because it may lead toa wide range of substituted compounds. From imidazole (Compound 15) onecan alkylate with 1,3-propanesultone to provide NHC precursor Compound16, or prior to that one can treat with an NHC to access the wealth ofdiazonium chemistry to provide Compound 17 in all of its forms.Solvolysis in water or alcoholic solvent may provide a phenol or arylether, and copper mediated Sandmeyer-type chemistry may afford cyano,nitro or halogenated aryl species. From imidazolium Compound 16Staudinger chemistry followed by aniline alkylation may provide Compound18, or traceless Staudinger-Bertozzi ligation may yield Compound 19.These substrates cover a range of Hammett values while also providing anadditional site of attachment to proteins, fluorophores, surfaces, etc.

Referring to FIG. 7B, regarding the role of intramolecular hydrogen bondacceptors/donors in reactivity, it may be possible to synthesize aseries of triazabutadienes with hydrogen bond donors that possess arange of pKa values (Compounds 20-22). In addition to H-bond donors, itmay be possible to synthesize a series of internal bases (Compounds23-25). It may be possible that that bases positioned near the N1nitrogen will favor protonation at N3 and thus make the triazabutadieneless stable to acidic media. These compounds are all synthetic targetsgiven a strategy of coupling with aryl azides. The delicatetriazabutadiene functional group is installed last under mildconditions. In addition to compounds that are designed toactivate/deactivate the N1 nitrogen, it may be possible to synthesize aseries of compounds where the N3 nitrogen in most likely to be affected(Compounds 26-28). An NHC with a hydrogen bond donor on a short arm wasmade. As in FIG. 7C, the synthesis of Compounds 26-28 from knownCompound 29 may start with either alkylation to a compound like Compound31 or reduction and protection to Compound 30 followed by alkylation toCompound 32. If the mesityl is absolutely essential for a desiredreactivity profile, a H-bond donor/acceptor may be inserted on a methylgroup in the ortho position of the mesityl ring.

Referring to FIG. 7D, regarding intramolecular trapping of diazoniumspecies, it may be possible to synthesize triazabutadienes with adjacentfunctional groups that will rapidly consume the diazonium species. Forexample, Compound 33 contains an aryl ring, positioned ortho to themasked diazonium. The synthesis may start from a diazo transfer reactionto convert aniline Compound 34 to an aryl azide. Coupling with Compound5c (see FIG. 6E) may complete the synthesis. It is possible thatfollowing diazonium unmasking an aromatic substitution reaction willoccur to provide benzocinnoline Compound 35. Because this reaction isintramolecular one might be able to use a non-activated ring, renderingthe ring electron rich. The methyl ether may serve as a site ofattachment to chemical cargos. A second type of intramolecular diazoniumtrap that could be employed is a beta keto ester that is also ortho tothe diazonium produced. Beta keto esters are known to react withdiazonium species through enol form, and can generate oxo-cinnolines,which are biologically active cores.

IV. Releasable Triazabutadienes (Pro-Triazabutadienes)

a. Pro-Triazabutadiene Synthesis and Mechanisms

The present invention also features triazabutadienes that may bereleased from pro-triazabutadienes (triazabutadiene precursors), e.g.,under appropriate conditions. The present invention also featurestriazabutadienes that are used to synthesize said pro-triazabutadienes.

Inventors have surprisingly discovered that carboxylation on N1 oftriazabutadienes reversibly yields stabilized or protected versions oftriazabutadienes, e.g., pro-triazabutadienes that are generally stablein acidic conditions. For example, carboxylation on N1 yielded apro-triazabutadiene molecule that is stable in concentrated HCl inmethanol; treating the pro-triazabutadiene molecule with NaOH inmethanol returned the original triazabutadiene molecule (which can thenbe degraded, e.g., in acidic conditions). Thus, the pro-triazabutadienemolecule may function as a means to protect triazabutadienes fromdegradation, e.g., under acidic conditions.

The present invention features pro-triazabutadiene molecules that underappropriate conditions (e.g., chemical conditions, enzymatic conditions,light, etc.) yield or release a triazabutadiene molecule. As usedherein, the terms “releasable triazabutadiene” and “pro-triazabutadiene”refer to molecules that comprise an inactive form of a triazabutadienemolecule (e.g., a protected version of a triazabutadiene) but can yieldor release an active triazabutadiene molecule upon appropriateconditions. The active triazabutadiene molecule could then go on torelease a diazonium species. That diazonium species could then reactwith a phenol (e.g., a tyrosine molecule), e.g., in a coupling reaction,or the diazonium species could self-immolate to release a phenol.

The conversion of a triazabutadiene molecule to a protectedtriazabutadiene (or pro-triazabutadiene), e.g., carboxylation on N1, isreversible. Without wishing to limit the present invention to any theoryor mechanism, it is believed that a mechanism that involves pushingelectrons onto the carbonyl carbon would result in cleavage of thecarbon-N1 nitrogen bond. And, there are triggered release processes thatcould undergo such an event. For example, some reactions are optimizedto lose carbon dioxide via carbarmic acids. It is possible that aquinone methide base linker may function well for triggered release aswell.

FIG. 8A shows a non-limiting example of a formula (Formula B) forreleasable triazabutadiene molecules (pro-triazabutadiene molecules) ofthe present invention. In some embodiments, pro-triazabutadienes maycomprise a formula according to Formula B. In some embodiments, Z²comprises a carboxyl group (N1 nitrogen is carboxylated). X¹ and Y¹ havebeen previously described. For example, in some embodiments, X¹comprises a mesityl group. In some embodiments, Y¹ comprises a mesitylgroup. In some embodiments, Z² (the carboxyl group) comprises CO₂Et. Thepresent invention is not limited to the aforementioned examples andformulas. For example, Z² may comprise a carboxyl group different fromCo2Et.

In some embodiments, the pro-triazabutadienes comprise atriazabutadiene, e.g., according to Formula B, wherein the N1 nitrogenof the triazabutadiene is modified such that the modifiedtriazabutadiene is more stabile at a particular pH as compared to theunmodified triazabutadiene. For example, in some embodiments, themodified triazabutadiene (pro-triazabutadiene) is more stabile at pH 5.5as compared to the unmodified triazabutadiene. In some embodiments, themodification comprises carboxylation (on the N1 nitrogen). The presentinvention is not limited to modifications comprising carboxylation onthe N1 nitrogen. In some embodiments, the modification comprises anortho-quinone methide linked on the N1 nitrogen.

Further, the present invention is not limited to the N1 carboxylationsdescribed herein. For example, other carbonyl variations or derivativesmay be used, e.g., the ethyl formate side group shown in Compound 58 ofFIG. 8B may be different, e.g., the carbonyl oxygen may be replaced withsulfur, one or both oxygen atoms may be replaced with nitrogen, anoxygen atom may be removed, etc.

In some embodiments, the triazabutadiene molecules that form thepro-triazabutadiene molecules are water-soluble. In some embodiments,the triazabutadiene molecules that form the pro-triazabutadienemolecules are not water-soluble.

To test reversibility of carboxylation on N1, triazabutadiene Compound57 was synthesized from Compound 58 and ethylchloroformate (see FIG.8B). It was observed that Compound 57 was stable to a 20% solution ofconcentrated HCl in methanol overnight (confirmed by NMR and in thepresence of resorcinol). Conversely, Compound 57 readily reacted withNaOH in methanol, returning the base-stable precursor Compound 58. Trueto mechanism, acidification of this solution with HCl rapidly yieldedthe benzene diazonium ion and subsequently reacted with the resorcinolpresent to provide the azobenzene product, Sudan Orange G. Thetriazabutadiene is a nucleophile and thus stable to biologicalnucleophiles, such as thiols, but it is possible that thiols will nowreact with electrophilic Compound 57 to provide side reactions (e.g.,undesirable side reactions). When Compound 57 was tested with two-foldexcess of β-mercaptoethanol (BME) in methanol, it was determined thatthe half-life was ˜20 hours. The reaction appeared to cleanly provide aneutral compound that has been reduced.

b. Triazabutadiene release

A variety of triggers may be employed to release the triazabutadienefrom the pro-triazabutadiene. The release of the triazabutadienemolecule may in some cases be selectively triggered. For example, insome embodiments, the trigger is an enzymatic trigger that selectivelyreacts with the protected triazabutadiene to deprotect the protectedtriazabutadiene, possibly allowing for aryl diazonium release.

FIG. 14A protection of a triazabutadiene to allow its selectiveactivation via an enzymatic trigger. A triazabutadiene is shown on theleft. The triazabutadiene may be converted to a protectedtriazabutadiene (middle molecule), e.g., pro-triazabutadiene. Theprotected triazabutadiene may feature protection at the N1 nitrogen tohelp prevent the rapid release of the aryl diazonium that may typicallyoccur if the triazabutadiene is not protected. The protectedtriazabutadiene may, upon an enzymatic trigger (right molecule), yieldan aryl diazonium species.

Enzymatic triggers may include but are not limited to phosphatases,sulfatases, esterases, nitroreductases, etc., or any enzyme that cleavesa bond. Such enzymes are well known to one of ordinary skill in the art.A triazabutadiene may be created or tailored to be cleavable by aparticular enzyme of choice.

In some embodiments, the compound used to protect the triazabutadiene(e.g., see first reaction of FIG. 14A) may be termed a protecting group.In some embodiments, the protecting group comprises a drug or pro-drug.In some embodiments, upon an enzymatic trigger, the drug or pro-drug isreleased. The protecting groups are not limited to drugs; anyappropriate molecule may be considered.

The present invention features a range of moieties that can be utilizedto trigger triazabutadiene release (FIG. 8C shows one example). Forexample, ortho-nitro derivatives (e.g., Compound 58b) may providephoto-caged moieties. The light used to un-cage (˜365 nm) may also speedup diazonium release. In addition to the photo-uncaged triazabutadienes,the present invention also features enzymatically-triggered derivatives.As an example, FIG. 8D shows β-lactamase substrate Compound 59. Thestrained ß-lactam ring is opened whereupon the nitrogen is now inconjugation with a π-system that can facilitate the release of a leavinggroup and yield Compound 60. The synthesis of this compound may followknown routes to Compound 61 (from commercially available Compound 62,see FIG. 8E) to the chloroformate precursor. These enzymes are stableand catalytically active throughout the pH 5.5-8 range (which may beideal for testing the stability of the activated triazabutadiene).

FIG. 8F shows a tetrahedral intermediate (see Key Intermediate). Thistetrahedral intermediate may be important for release strategies. Forexample, the loss of carbon dioxide from the intermediate yields theoriginal triazabutadiene (e.g., see Compound A in FIG. 8F).

FIG. 8G shows synthesis of several protected triazabutadienes withvarying electronic properties on the aryl ring. The inductively electrondonating p-methyl substituent, 8, slowed the rate of deprotectionmoderately. Without wishing to limit the present invention to any theoryor mechanism, it is believed that this can be rationalized by a modelwhereby the carbonyl is stabilized by being more electron rich and thusless prone to nucleophilic attack. The p-methoxy substituted compound.9, also slowed hydrolysis, so much so that the rate could not bedetermined due to the error of the analysis. Interestingly, both theinductively electron withdrawing p-trifluoromethyl compound, 10 andresonance withdrawing p-nitro substituted 11 slowed hydrolysis, with theinductive effect playing a much larger role. Only a small change in ratewas observed moving from ethyl 7 to neopentyl 12 and i-propyl 13 (forthe alkyl group appended to the carbamate).

In addition to rendering the triazabutadiene moiety more compatible withprotein bioconjugation strategies to modify proteins of interest, theacid-stabilization may make the triazabutadiene compatible with mosttraditional Boc-strategies of solid-phase peptide synthesis. A protectedtriazabutadiene bearing a t-butyl ester was synthesized (see FIG. 8H,FIG. 8I). The acid-labile ester was removed using trifluoroacetic acid(TFA) in dichloromethane. Following removal of the ester the carbamateprotection was removed under basic conditions and finally the resultingtriazabutadiene was treated with acid and resorcinol to provide anazo-benzene product.

FIG. 9 shows analyses of various protected triazabutadienes. Forexample, FIG. 9A shows synthesis of protected triazabutadienes and theirprospective yields using various chloroformate reagents. FIG. 9B shows ahydrolysis reaction of protected triazabutadienes where R═—CH₂CH₃ inbuffer. The reaction is assumed to be second order with the addition ofhydroxide to be the rate-determining step. FIG. 9C shows a decrease inabsorbance of the protected triazabutadiene where R═—CH₂CH₃ as thehydrolysis reaction proceeds over time. Compound was in a concentrationof 31 μM in 25 mM sodium borate buffer of pH 10 with scans every 60seconds. FIG. 9D shows the concentration (μM) of protectedtriazabutadiene where R═—CH₂CH₃ vs. time (min). Scans were obtainedevery 60 seconds. (Note the initial concentration of compound was 31 μMin buffers of various pH ranges. Buffers pH 8, 9, and 10:25 mM sodiumborate; buffer pH 7:100 mM phosphate; buffer pH 2:200 mM KCl buffer.)FIG. 9E shows a graph of ln(abs) vs. time (s) to obtain K_(obs) underpseudo-first order reaction conditions: a plot of K_(obs) vs. hydroxideconcentration (M) to obtain the second order rate constant (k₂). Thechart shows various protected triazabutadienes with their correspondingsecond order rate constants (k₃).

In some embodiments, the protected triazabutadiene is protected with aphotocleavable protecting group. For example, FIG. 9F shows synthesis ofa protected triazabutadiene with a photocleavable protecting group. FIG.9G shows a photocleavable protected triazabutadiene exposed to 365 nmlight to yield a regular triazabutadiene. In some embodiments, theprotected triazabutadiene is not susceptible to cleavage using acids orbases. In some embodiments, the protected triazabutadiene isphotocleavable (see FIG. 9H as an example of a photocleavable protectedtriazabutadiene that is not susceptible to cleavage using acids orbases).

The present invention also features releasable triazabutadienes that canbe appended to proteins, e.g., an azide-containing compound.

The releasable triazabutadiene molecules may provide opportunities forselective diazonium delivery as biochemical probes. The releasabletriazabutadiene molecules may provide for spatially selective release ofaryl diazonium ions. The reactivity may be generalized to be broadlyapplicable and triggered. For example, by parlaying the releasechemistry into amide bonds it may be possible to target proteases. And,endosomal proteases that viruses and other pathogens encounter uponentry should facilitate cleavage. If the caged triazabutadiene compoundsare not substrates for the enzyme then a quinone-methide strategy may beimplemented.

c. Applications for Protected/Pro-Triazabutadiene Molecules

As previously discussed, a variety of triggers (e.g., enzymaticcleavage, light, base, etc.) may be employed to release thetriazabutadiene from the pro-triazabutadiene. The release of thetriazabutadiene molecule may in some cases be selectively triggered.Thus, the pro-triazabutadiene molecules may allow for use in two-stepdrug release systems or other cargo release systems. Or, thepro-triazabutadiene molecules may provide for an easier means of proteinmodification, e.g., because the molecules can be worked with in a widerrange of pHs.

Without wishing to limit the present invention to any theory ormechanism, it is believed that the increased stability of thepro-triazabutadiene molecules in acidic environments may provide forenhanced processes of self-assembling monolayers for the production ofbiosensors. For example, the pro-triazabutadiene may be protected untilit is in the presence of any given enzyme, and upon that trigger, thetriazabutadiene molecule can be released, which can then go on to form adiazonium species, which can couple with particles such asnanoparticles, polymers, and biomacromolecules. For example, in someembodiments, the molecules of the present invention can be used to helpcouple target compounds to a sensor (e.g., see U.S. Pat. No. 8,668,978,the disclosure of which is incorporated herein in its entirety).

V. Applications and Methods of Use of Triazabutadienes

The triazabutadiene molecules of the present invention may be utilizedfor a variety of purposes. For example, in some embodiments, thetriazabutadiene molecules of the present invention are utilized for achemoselectively-cleavable linkage for use in biological/complexsettings where rapid, dean cleavage is of interest. In some embodiments,the triazabutadiene molecules are used for systems including but notlimited to drug delivery systems, protein-protein interaction systems,pH environment detection systems, etc. Applications of thesetriazabutadienes may fall under one (or more) categories of reactivity.

a. Diazonium Coupling Applications

Regarding diazonium coupling, the triazabutadiene molecules may be usedfor applications involving pH-dependent protein coupling. Generalexamples involve methods for detecting protein-protein proximity orprotein-protein interactions (in a sample). In some embodiments, themethod comprises providing a first protein, wherein the first protein isconjugated with a triazabutadiene molecule according to the presentinvention. The first protein may be introduced to a sample. In someembodiments, the triazabutadiene molecule encounters a low pH in thesample; in some embodiments, acid is added to the sample to lower the pHappropriately. As previously discussed, in the low pH environment, thetriazabutadiene molecule undergoes the irreversible reaction yieldingthe diazonium species and the cyclic guanidine species. As previouslydiscussed, the diazonium species is adapted to react with a phenolgroup; thus if there is a nearby protein with a tyrosine residue, thediazonium species may react with it yielding an azobenzene product(often colored, e.g., the dye Sudan Orange G is an azobenzene-containingdye) that is visually distinct from the triazabutadiene molecule and thediazonium species. As such, detection of the azo dye (e.g., SudanOrange) may be indicative of proximity or interaction of the firstprotein and the second protein. Thus, in some embodiments, the methodcomprises adding a second protein to the sample, wherein a tyrosine ofthe second protein may react with the diazonium species. In someembodiments, the second protein is already in the sample. In someembodiments, a tyrosine or phenol species conjugated to the secondprotein.

In some embodiments, the method comprises introducing to the sample afirst antibody specific for a first protein, wherein the first antibodyis conjugated with a triazabutadiene molecule according to the presentinvention. In some embodiments, the method comprises introducing to thesample a second antibody specific for a second protein. In someembodiments, the second antibody comprises a tyrosine. In someembodiments, the second antibody is conjugated with a phenol species. Insome embodiments, the method comprises introducing an acid to the sampleto appropriately lower the pH of the sample. As previously discussed, inthe low pH environment, the triazabutadiene molecule undergoes theirreversible reaction yielding the diazonium species and the cyclicguanidine species. As previously discussed, the diazonium species isadapted to react with a phenol group; thus if the phenol species isnearby, the diazonium species may react with it yielding an azo dye thatis visually distinct from the triazabutadiene molecule and the diazoniumspecies. As such, detection of the azo dye may be indicative ofproximity or interaction of the first protein and the second protein.

As a more specific example, the acid-labile reactivity oftriazabutadienes may be used to assist in work deducing interactionpartners between a virus and endosomally localized host proteins. Uponendosomal acidification a viral-bound diazonium species may be unmaskedand this may go on to react with Tyr-containing proteins that areassociating with the virus. It is possible that this system could beused to trap an interaction that is relevant at a key point of viralentry, e.g., the fusion of membranes. Herein are non-limiting examplesof synthesis of compounds that may be used in such systems, e.g., formodifying the viral surface. Lysine-reactive probes may be used tomodify the surface of viral proteins. Referring to FIG. 10A, bysynthesizing triazabutadiene Compound 36 bearing an N-hydroxysuccinimide(NHS) ester it may be possible to couple the compound to one of manyreactive Lys on the surface of the virus. As previously discussed, atriazabutadiene molecule may be attached to a viral protein (e.g., apurified viral protein). Then, a system such as a cell line (e.g.,mosquito cell line, human cell line, or even mosquitoes themselves) maybe infected with the viral protein. The infected system can be treatedappropriately. The azo dye may “label” any proteins that interact withor are nearby the viral protein (in the low pH environment). The presentinvention is not limited to this example.

Lys-NHS conjugation chemistry may work well on the basic side ofneutral, which may be beneficial for pH sensitive probes. Referring toFIG. 10A and FIG. 10B, Compound 36 may be made in a straightforwardfashion from NHC precursor Compound 5c (see FIG. 6E) and an aryl azide.It is possible that the steric congestion about the NHC may favor theunencumbered azide over the potentially reactive NHS ester. If the NHSester presents a problem during the synthesis it is possible to go intothe reaction with a carboxylate instead and follow that by a couplingwith N-hydroxysuccinimide. If electronically coupling the NHS ester tothe aryl system is detrimental to reactivity it is possible to considerinserting an alkyl or, if needed for additional solubility, polyethyleneglycol (PEG) linker. As an example, referring to FIG. 10B, a monoclonalantibody (e.g., mouse anti-biotin) may be modified with Compound 36.Once the surface is decorated with triazabutadienes, the extent oflabeling may be quantified by coupling to resorcinol (or otherappropriate alternative) in a low pH solution and the extent ofmodification may be analyzed by mass spectrometry. This may show thenumber of reactive triazabutadienes. Following this analysis, afluorescent goat anti-mouse secondary antibody may be added, and then agel-shift assay may be used to show that the two antibodies arecovalently linked in a pH dependent manner.

c. Triazabutadiene Probes for Protein Modification

The present invention also features a lysine-reactiveN-hydroxysuccinimide (NHS) modified triazabutadiene, e.g., Compound 68(see FIG. 11A). This was synthesized from bismestiyl imidazolium, e.g.,Compound 69 (FIG. 11B). A series of derivatives is shown in FIG. 11C.Several variants of Compound 68 may be synthesized and tested. Forexample, Compound 70 with a sulfonate-containing NHS ester may provide aprotein modified identically to using Compound 8, but it is may besoluble at higher concentrations, which may enable more rapid labelingof dilute protein samples (such as viral samples). Anothersulfonate-containing derivative, Compound 71, may have the effect ofadding a negative charge to the surface of the protein that it modifies.A third probe that may be synthesized, Compound 72, contains a cysteine(thiol) reactive maleimide, which may offer a greater degree ofselectivity due to the low abundance of surface exposed thiols. Thisprobe may be used in conjunction with proteins that have been mutated topossess a solvent expose cysteine at positions of interest. The N-linkedamide probe, Compound 73, may be used to look at those electroniceffects in the context of a complex biological sample.

In the absence of a protein cross-linking event, there may be an aryldiazonium, which decomposes to a phenol and remains bound to the lysine.This phenol is likely prone to redox chemistry and as such represents anavenue for complexity during proteomic analysis. A self-immolatingtriazabutadiene has been designed to circumvent these pitfalls.Referring to FIG. 12, triazabutadiene Compound 80 provides an aryldiazonium (Compound 81), which can go on to react with locally availabletyrosine residues, or decompose to phenol Compound 82 if none areavailable. This phenol will further degrade via quinone-methidechemistry to extrude carbon dioxide and return an unaltered lysineresidue.

d. Diazonium Degradation for Cargo or Drug Release

In some embodiments, the triazabutadiene molecules of the presentinvention may be used in applications involving diazonium degradation torelease cargo or drugs. For example, a group of applications takesadvantage of the solvolysis of diazonium salts to produce phenolicbyproducts. The degradation of diazonium salts to phenols, via arylcations, is a first-order process that is not pH dependent in thephysiological range of pHs. The half-life of this first order processdepends on substitution on the aryl ring; the rate for benzenediazoniumis ˜4 hours. Indeed, the product of this degradation and subsequentazo-dye formation was observed if resorcinol is not put into thebuffered NMR experiments.

In some embodiments, the acid-dependent instability of thetriazabutadiene molecule may allow for a drug or cargo molecule to bedeposited at a desired location and time (e.g., the reaction can becontrolled and initiated at a desired time and location). As such, thepresent invention also features methods of delivering a drug (or a cargocompound) to a subject. In some embodiments, the method comprisesproviding a triazabutadiene molecule according to the present invention,conjugating a drug (or cargo compound) to the triazabutadiene molecule;and administering the conjugate (the drug/cargo-triazabutadieneconjugate) to the subject. In some embodiments, the method comprisesproviding a triazabutadiene molecule according to the present inventionwherein the triazabutadiene molecule comprises the drug (or cargocompound); and administering the triazabutadiene molecule to thesubject. In some embodiments, the diazonium species of thetriazabutadiene molecule is part of the drug (or cargo compound). Insome embodiments, the drug (or cargo compound) is formed when thediazonium species reacts to a phenol species. In some embodiments, thedrug is an anti-cancer drug.

The drug (or cargo compound) is not limited to an anti-cancer drug. Anyappropriate drug for any appropriate condition may be considered.Likewise, the triazabutadiene molecules may be incorporated intodrug/cargo-delivery systems for conditions including but not limited tocancer or other conditions associated with low pH states (e.g.,gastrointestinal conditions, sepsis, ketoacidosis, etc.). Non-limitingexamples of drugs (e.g., drugs that have a phenolic functional group,which may be masked as prodrugs) include: Abarelix, Alvimopan,Amoxicillin, Acetaminophen, Arformoterol, Cefadroxil, Cefpiramide,Cefprozil, Clomocycline, Daunorubicin, Dezocine, Epinephrine,Cetrolrelix, Etoposide, Crofelemer, Ezetimibe, Idarubicin, Ivacaftor,Hexachlorophene, Labetalol, Lanreotide, Levodopa, Caspofungin,Butorphanol, Buprenorphine, Dextrothyroxine, Doxorubicin, Dopamine,Dobutamine, Demeclocycline, Diflunisal, Dienestrol, Diethylstilbestrol,Doxycydine, Entacapone, Arbutamine, Apomorphine, Baisalazide, Capsaicin,Epirubicin, Esterified Estrogens, Estradiol Valerate, Estrone,Estradiol, Ethinyl Estradiol, Fulvestrant, Goserelin, Fluorescein,Indacaterol, Levosalbutamol, Levothyroxine, Liothyronine, Lymecycline,Mitoxantrone, Monobenzone, Morphine, Masoprocol, Mycophenolic Acid,Phenylephrine, Phentolamine, Oxytetracycline, Rifaximin, Rifapentine,Oxymetazoline, Raloxifene, Tolcapone, Terbutaline, Tetracycline,Mesalamine, Metaraminol, Methyldopa, Minocycline, Nabilone, Nalbuphine,Nelfinavir, Propofol, Rotigotine, Ritodrine, Salbutamol, Sulfasalazine,Salmeterol, Tapentadol, Tigecycline, Tolterodine, Teniposide,Telavancin, Topotecan, Triptorelin, Tubacurarine, Valrubicin,Vancomycin, etc.

In some embodiments, drug delivery systems featuring triazabutadienemolecules may be enhanced with other reactions, e.g., enzymaticreactions. Such additional reactions may help provide appropriatespecificity of the drug delivery system or appropriate timing to thedrug delivery system.

Referring to FIG. 13, the triazabutadiene molecules of the presentinvention may be used for applications involving benzoquinone methides,e.g., it may be possible to synthesize derivatives that can undergoelimination via para-quinone methide chemistry (see FIG. 13A). Referringto FIG. 13A, after acidification, triazabutadiene Compound 41 maydecompose to diazonium salt (Compound 42). This reactive species maydecompose to a phenol (Compound 43), which itself decomposes to aquinone methide and may liberate the cargo molecule (Compound 44). Itmay be possible to modify the electronic properties of the central ringin order to influence the rates at each step. This system is may beuseful for these modifications because none of them are expected toaffect the cargo. The azide-coupling chemistry may render this amenableto wide variety of chemical cargos. In a biological context thesecompounds may be able to release their desired cargo upon entry into theendosome, or upon exposure to non-virally relevant acidic environmentssuch as in proximity to cancerous tumors. This type of attachmentchemistry may be utilized as a method for drug or detection delivery,and may have an added level of specificity if the system was deliveredto a desired location using an antibody or aptamer.

In some embodiments, Z¹ (see FIG. 1, FIG. 13C) is a prodrug comprising aphenolic functional group, wherein the phenolic group is masked as atriazylidene moiety. An example of how a prodrug is released (e.g., inan acidic environment, e.g., in a patient) is illustrated in FIG. 13B.Without wishing to limit the present invention to any theory ormechanism, it is believed that all drugs, such as those approved by theU.S. Food and Drug Administration, that have a phenolic functional groupmay be masked as a triazylidene moiety.

Referring to FIG. 13B, Compound C is a stimulant that is produced byGlaxo Smith-Kline Beecham pharmaceutical company. The phenolic group ofCompound C can be converted to an azide group, e.g., by displacement ofthe hydroxyl group with an azide group. In some embodiments, thephenolic group is first converted to a suitable leaving group beforesubjecting to a nucleophilic displacement reaction with an azide group.The resulting azide Compound A is then reacted with3-(3-mesityl-2-(phenyltriaz-2-en-1-ylidene)-2,3-dihydro-1H-imidazol-1-yl) propane-1-sulfonate to produce triazylideneCompound B. In some embodiments, when Compound B is administered to apatient (e.g., orally or intravenously), the acidic environment of thepatient's gastrointestinal tract (if administered orally) or patient'sblood plasma (when administered intravenously) decomposes it to generatea corresponding diazonium compound regenerates the phenolic group asillustrated in FIG. 13B. By converting the phenolic group (e.g., thehydroxyl group that is attached to a phenyl ring) to an azide, oneskilled in the art having read the present application can readilyconvert the phenol compound to a triazylidene compound of the invention.Thus, the triazylidene moiety serves as a masking group for a phenolicfunctional group.

The present invention also features a method for administering a drugcomprising a phenolic function group to a subject in need of such a drugadministration. In some embodiments, the method comprises converting adrug comprising a phenolic-functional group to a prodrug, wherein saidprodrug comprises an acid labile triazylidene moiety; and administeringsaid prodrug to a subject in need of such a drug administration. In someembodiments, the triazylidene compound may also comprise a watersolubility conferring moiety and/or Y¹ functional group defined in FIG.1.

The present invention also features a method of converting a drugcomprising a phenolic-function group to an acid labile prodrug. In someembodiments, the phenolic-functional group is converted to an azidegroup. The azide functional group may then be reacted with a carbene toproduce an acid labile prodrug comprising a triazylidene moiety (seeFIG. 13D).

d. Other Applications

In some embodiments, a triazabutadiene molecule is conjugated to anothermolecule (a conjugate molecule), e.g., a protein (e.g., an amino acidsuch as but not limited to lysine), a lipid, or other appropriatemolecule. In some embodiments, the diazonium species part of thetriazabutadiene molecule is conjugated to the conjugate molecule. Insome embodiments, the cyclic guanidine species part of thetriazabutadiene molecule is conjugated to the conjugate molecule. Insome embodiments, the triazabutadiene molecule is attached to theconjugate molecule via a linker. Linkers are well known to one ofordinary skill in the art and may include (but are not limited to) apolyether linkers such as polyethylene glycol linkers. In someembodiments, the conjugate molecule to which the triazabutadienemolecule is conjugated comprises an antibody or a fragment thereof. Insome embodiments, the conjugate molecule to which the triazabutadienemolecule is conjugated comprises a viral protein.

In some embodiments, the triazabutadiene molecules of the presentinvention are used for pull-down studies wherein a biomolecule orprotein of interest is attached to one side and the other side isappended to something such as but not limited to a small molecule (e.g.,hapten such as biotin) or compound. Using biotin as an example, thebiomolecule or protein of interest can be pulled down using an avidinbead (which binds strongly to the biotin) and thoroughly washed. Thismay be useful for protein enrichment. The biomolecule or protein ofinterest may then be cleaved from the avidin bead by means of reductivecleavage of the triazabutadiene that holds them together. The presentinvention is not limited to these components, for example thisapplication could also feature the use of a probe (e.g., fluorescent orotherwise) attached to an antibody used to interrogate a complex sample.

In some embodiments, reductive cleavage of triazabutadiene molecules mayalso be used to cleave unreacted triazabutadienes that did not undergodiazonium formation/reaction chemistry that is associated with a drop inpH (or other mechanism) as described above (a sort of quench for the pHchemistry).

As previously discussed, the diazonium species can react with a phenolspecies such as resorcinol or other appropriate phenol species. In someembodiments, a phenol species or resorcinol species is conjugated to aprotein, e.g., a protein different from the protein to which thetriazabutadiene molecule is conjugated, a protein that is the sameprotein to which the triazabutadiene molecule is conjugated, etc. Insome embodiments, the resorcinol species or phenol species that thediazonium species reacts with is the phenol functional group of atyrosine residue.

The present invention also features a method of detecting an environmenthaving a low pH. In some embodiments, the method comprises providing asample (e.g., tissue sample, cell sample, any appropriate sample) andintroducing a triazabutadiene molecule according to the presentinvention to the sample. An environment having a low pH (a low pHappropriate for the triazabutadiene molecule) causes the triazabutadienemolecule to break down into a diazonium species and a cyclic guanidinespecies. Since the diazonium species is visually distinct from thetriazabutadiene, visualization of the diazonium species is indicative ofthe low pH environment. In some embodiments, the method furthercomprises introducing a resorcinol species or a phenol species to thesample. The resorcinol species or phenol species may react with thediazonium species to form an azo dye. Since the azo dye is visuallydistinct from the diazonium species and the triazabutadiene species,detection of the diazonium species and/or the azo dye would beindicative of the low pH environment.

The present invention is not limited to the methods and uses describedherein. For example, in some embodiments, the triazabutadiene moleculesare used as reagents in buffers for various chemical or biochemicalassays (e.g., immunohistochemistry assays, in situ hybridization assays,protein assays such as western blots, ELISAs, etc.).

The triazabutadiene molecules may function as masked compounds that,when exposed to water, form reaction products that form covalent bondswith surfaces containing phenols. In some embodiments, thetriazabutadiene molecule (or a diazonium species) is conjugated to amolecule other than a glass or plastic as described above. In someembodiments, the triazabutadiene molecule (or a diazonium species) isconjugated to a surface via a linker. Linkers are well known to one ofordinary skill in the art and may include (but are not limited to) apolyether linkers such as polyethylene glycol linkers.

In some embodiments, a triazabutadiene molecule is bonded to a surface.Surfaces may include but are not limited to glass, plastic, abiomaterial, or any other appropriate surface, Non-limiting examples ofmaterials also include Tufnol materials such as phenolic cottonlaminated plastics, phenolic paper laminated plastics, etc., a phenolformaldehyde resin such as bakelite (or baekelite), etc.

The present invention is not limited to the methods and uses describedherein. For example, in some embodiments, the molecules herein are usedas reagents in buffers for various chemical or biochemical assays (e.g.,immunohistochemistry assays, in situ hybridization assays, proteinassays such as western blots, ELISAs, etc.).

The disclosures of the following documents are incorporated in theirentirety by reference herein: U.S. Pat. No. 8,617,827; U.S. Pat.Application No. 2009/0048222; U.S. Pat. No. 3,591,575. U.S. Pat. Nos.3,607,542; 4,107,353; WO Pat. No. 2008090554; U.S. Pat. No. 4,218,279;U.S. Pat. App. No. 2009/0286308; U.S. Pat. Nos. 4,356,050; 8,603,451;5,856,373; 4,602,073; 3,959,210. The disclosures of the followingpublications are incorporated in their entirety by reference herein:Kimani and Jewett, 2015, Angewandte Chemie International Edition (DOI:10.1002/anie.201411277—Online ahead of print). Zhong et al., 2014,Nature Nanotechnology 9, 858-866; Stewart et al., 2011, J Polym Sci BPolym Phys 49(11):757-771; Poulsen et al., 2014, Biofouling30(4):513-23; Stewart, 2011, Appl Microbiol Biotechnol 89(1):27-33;Stewart et al., 2011, Adv Colloid Interface Sci 167(1-2):85-93;Hennebert et al., 2015, Interface Focus 5(1):2014.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference cited in the presentapplication is incorporated herein by reference in its entirety.

Although there has been shown and described the preferred embodiment ofthe present invention, it will be readily apparent to those skilled inthe art that modifications may be made thereto which do not exceed thescope of the appended claims. Therefore, the scope of the invention isonly to be limited by the following claims. Reference numbers recited inthe claims are exemplary and for ease of review by the patent officeonly, and are not limiting in any way. In some embodiments, the figurespresented in this patent application are drawn to scale, including theangles, ratios of dimensions, etc. In some embodiments, the figures arerepresentative only and the claims are not limited by the dimensions ofthe figures. In some embodiments, descriptions of the inventionsdescribed herein using the phrase “comprising” includes embodiments thatcould be described as “consisting of”, and as such the writtendescription requirement for claiming one or more embodiments of thepresent invention using the phrase “consisting of” is met.

The reference numbers recited in the below claims are solely for ease ofexamination of this patent application, and are exemplary, and are notintended in any way to limit the scope of the claims to the particularfeatures having the corresponding reference numbers in the drawings.

What is claimed is:
 1. A compound of Formula B:

wherein: A is —N—; D is —H; E is —H; X¹ is an aryl, an alkyl, acarboxylic acid, an alcohol, or an amine; Y¹ is an aryl, an alkyl, acarboxylic acid, an alcohol, or an amine; Z¹ is an aryl; and Z² is amoiety comprising a carbonyl group; wherein the compound releases atriazabutadiene molecule and a derivative of Z² when subjected to atrigger that results in deprotection of N1 nitrogen.
 2. The compound ofclaim 1, wherein X¹ and Y¹ are both alkyl.
 3. The compound of claim 1,wherein X¹ is methyl.
 4. The compound of claim 1, wherein Y¹ is methyl.5. The compound of claim 1, wherein X¹ and Y¹ are methyl.
 6. Thecompound of claim 1, wherein Y¹ is t-butyl.
 7. The compound of claim 1,wherein X¹ and Y¹ are both t-butyl.
 8. A method of selectivelyactivating a triazabutadiene, said method comprising introducing atrigger to a compound according to Formula B:

wherein: A is —N—; D is —H; E is —H; X¹ is an aryl, an alkyl, acarboxylic acid, an alcohol, or an amine; Y¹ is an aryl, an alkyl, acarboxylic acid, an alcohol, or an amine; Z¹ is an aryl; and Z² is amoiety comprising a carbonyl group; wherein the trigger results indeprotection of N1 nitrogen to cause the compound to yield an activetriazabutadiene molecule and a derivative of Z².
 9. The method of claim8, wherein the trigger is basic conditions.
 10. The method of claim 8,wherein the trigger is an enzyme.
 11. The method of claim 8, wherein thetrigger is a redox environment.
 12. The method of claim 8, wherein thetrigger is a redox environment inside a cell.
 13. The method of claim 8,wherein the trigger is light.
 14. The method of claim 8, wherein thecompound of Formula B is stable in acidic conditions having a pH of 6.0or less.
 15. The method of claim 8, wherein the derivative of Z² is adrug or a pro-drug.
 16. The method of claim 8, wherein the activetriazabutadiene molecule yields an aryl diazonium species.
 17. Themethod of claim 8, wherein X¹ and Y¹ are both alkyl.
 18. The method ofclaim 8, wherein X¹ or Y¹ is methyl.
 19. The method of claim 8, whereinX¹ and Y¹ are both methyl.
 20. The method of claim 8, wherein X¹, Y¹, orboth X¹ and Y¹ are t-butyl.